Evaluation of a novel monoclonal antibody mAb109 for noninvasive molecular imaging of lung carcinoma

Objectives Here we report the evaluation of a novel monoclonal antibody mAb109 which is produced by cell fusion techniques as a promising lung carcinoma imaging agent. The mAb109 is for the first time modified by near-infrared fluorescent (NIRF) dye and PET radionuclide, respectively. The resulted optical and PET immuno-probes have been further evaluated in mice bearing non-small lung carcinoma A549tumors.

Methods mAb109 and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) conjugated antibody (NOTA-mAb109) were prepared and labeled with Cy5.5 and 64Cu,respectively. Cy5.5 and 64Cu-NOTA conjugated IgG were also prepared as controls that showed no specific binding to tumor. The tumor cell binding affinities of Cy5.5-mAb109 and64Cu-NOTA-mAb109 were tested in A549 cells. NIRF/Micro-PET imaging of four probes were evaluated in A549 lung carcinoma xenografts in athymic nude mice (n=4 per group). Optical images were acquired until 16 days after injection of Cy5.5-mAb109. And micro-PET/CT images were acquired until 3 days. Mice were euthanized after the last PET/CT imaging session, and the biodistribution of the 64Cu-probes was performed. The autoradiography of 64Cu-antibody in tumor tissues was also tested.

Results The Cy5.5-mAb109 was synthesized withdye to mAbratio of 1.38. The uptake of the NIRF probe to A549 cellswas blocked by excess mAb109 as detected by fluorescence microscopy. For in vivo NIRF imaging study, the probe showedhigh and specific tumor contrast and retention. The A549 tumor to background ratio was 3.5at 16 days post-injection(p.i.). Interesting, the A549 tumor was barely visible for the blocking group (Cy5.5-mAb109 plus 100 µg of mAb109) and Cy5.5-IgG. The ⁶⁴Cu-NOTA-mAb109 was synthesized with a specific activity of 0.74 MBq/μg of antibody. The binding affinity of ⁶⁴Cu-NOTA-mAb109 with A549 cells was determined to be 29.64 nM. Biodistribution study revealed that⁶⁴Cu-NOTA-mAb159 displayed prominent tumor accumulation from 2 hours to 60 h (the tumor uptake was 1.84±0.41 and 9.34±0.67%ID/g, respectively). At the same time, the tumor uptake was 2.29±0.47 and2.68±0.69%ID/g in the blocking and ⁶⁴Cu-NOTA-IgG groups, respectively.In vivo Micro-PET images of ⁶⁴Cu-NOTA-mAb109 also displayed significantly higher (p<0.05) tumor accumulation (ROI analysis showed it up to 9.5%ID/g) than that of blocked and ⁶⁴Cu-NOTA-IgG. The autoradiography of ⁶⁴Cu-antibody also confirmed this finding.⁶⁴Cu-NOTA-mAb109 had a higher accumulationin A549 tumor. In contrast, ⁶⁴Cu-NOTA-IgG and the blocked groups had significant lower A549 tumor accumulation (p<0.05).

Conclusions Our study demonstrates that mAb109 antibody can serve as a new platform for development of novel agents for lung carcinoma non-invasive imaging. The resulted Cy5.5-mAb109 and ⁶⁴Cu-NOTA-mAb109 showhighspecificity and favorable imaging properties for imaging of non-small lung cancer A549 tumor models. Acknowledgements: This work was supported, in part, by the Office of Science (BER), U.S. Department of Energy (DE-SC0008397). And by the National Natural ScienceFoundation of China (81172083, 81371592, 81401467, 81501519).